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Abstract Detection and remediation of stress in crops is vital to ensure agricultural productivity. Conventional forms of assessing stress in plants are limited by feasibility, delayed phenotypic responses, inadequate specificity, and lack of sensitivity during initial phases of stress. While mass spectrometry is remarkably precise and achieves high-resolution, complex samples, such as plant tissues, require time-consuming and biased depletion strategies to effectively identify low-abundant stress biomarkers. Here, we bypassed these reduction methods via a nano-omics approach, where gold nanoparticles were used to enrich time- and temperature-dependent stress-related proteins through biomolecular corona formation that were subsequently analyzed by ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). This nano-omic approach was more effective than a conventional proteomic analysis using UHPLC- MS/MS for resolving biotic-stress induced responses at early stages of pathogen infection inArabidopsis thaliana, well before the development of visible phenotypic symptoms, as well as in distal tissues of pathogen infected plants at early timepoints. The enhanced sensitivity of this nano-omic approach enables the identification of stress-related proteins at early critical timepoints, providing a more nuanced understanding of plant-pathogen interactions that can be leveraged for the development of early intervention strategies for sustainable agriculture. 

Abstract Lipid nanoparticles (LNPs) are the most clinically advanced nonviral RNA-delivery vehicles, though challenges remain in fully understanding how LNPs interact with biological systems.In vivo, proteins form an associated corona on LNPs that redefines their physicochemical properties and influences delivery outcomes. Despite its importance, the LNP protein corona is challenging to study owing to the technical difficulty of selectively recovering soft nanoparticles from biological samples. Herein, we developed a quantitative, label-free mass spectrometry-based proteomics approach to characterize the protein corona on LNPs. Critically, this protein corona isolation workflow avoids artifacts introduced by the presence of endogenous nanoparticles in human biofluids. We applied continuous density gradient ultracentrifugation for protein-LNP complex isolation, with mass spectrometry for protein identification normalized to protein composition in the biofluid alone. With this approach, we quantify proteins consistently enriched in the LNP corona including vitronectin, C-reactive protein, and alpha-2-macroglobulin. We explore the impact of these corona proteins on cell uptake and mRNA expression in HepG2 human liver cells, and find that, surprisingly, increased levels of cell uptake do not correlate with increased mRNA expression in part likely due to protein corona-induced lysosomal trafficking of LNPs. Our results underscore the need to consider the protein corona in the design of LNP-based therapeutics. Abstract Figure 

Abstract Understanding the interaction between biological structures and nanoscale technologies, dubbed the nano-bio interface, is required for successful development of safe and efficient nanomedicine products. The lack of a universal reporting system and decentralized methodologies for nanomaterial characterization have resulted in a low degree of reliability and reproducibility in the nanomedicine literature. As such, there is a strong need to establish a characterization system to support the reproducibility of nanoscience data particularly for studies seeking clinical translation. Here, we discuss the existing key standards for addressing robust characterization of nanomaterials based on their intended use in medical devices or as pharmaceuticals. We also discuss the challenges surrounding implementation of such standard protocols and their implication for translation of nanotechnology into clinical practice. We, however, emphasize that practical implementation of standard protocols in experimental laboratories requires long-term planning through integration of stakeholders including institutions and funding agencies. 

Abstract Robust characterization of the protein corona—the layer of proteins that spontaneously forms on the surface of nanoparticles immersed in biological fluids—is vital for prediction of the safety, biodistribution, and diagnostic/therapeutic efficacy of nanomedicines. Protein corona identity and abundance characterization is entirely dependent on liquid chromatography coupled to mass spectroscopy (LC-MS/MS), though the variability of this technique for the purpose of protein corona characterization remains poorly understood. Here we investigate the variability of LC-MS/MS workflows in analysis of identical aliquots of protein coronas by sending them to different proteomics core-facilities and analyzing the retrieved datasets. While the shared data between the cores correlate well, there is considerable heterogeneity in the data retrieved from different cores. Specifically, out of 4022 identified unique proteins, only 73 (1.8%) are shared across the core facilities providing semiquantitative analysis. These findings suggest that protein corona datasets cannot be easily compared across independent studies and more broadly compromise the interpretation of protein corona research, with implications in biomarker discovery as well as the safety and efficacy of our nanoscale biotechnologies.